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I recently started a liquid mycellium culture experiment and am giving a detailed account.

I would like to hear any input/opinions/reccomendations on methods etc..

I recently took a rhizomorphic section of EQ mycellium and added it to 10 mL of L-broth (1% tryptone peptone, 0.5% yeast extract, 0.5% NaCl, pH approx 7 ) medium. After vortexing to break up the mycellium, the culture tube was incubated at 37 C ( 98.6 F). After a week of no visible growth, and I believed that I had thermally killed the mycellium. Midway through week two, the little bastard took off! By the end of week two, strong mycellial growth was visible in the culture tube (entire bottom of culture tube white with mycellium).

I decided to innoculate some autoclaved honey water medium (wt % unknown, pH unknown, I was lazy that day) with the mycellial culture.

100 mL of autoclaved/UV-C irradiated honey water was double filtered through 0.2 um sterile membrane filters; the solution was divided into three 30 mL aliquots. Approximately 5 mL of the mycellium containing L-broth medium was added to two of the three honey water samples. All three samples were sealed (rubber septa), wrapped in foil, and placed in the incubator (37 C, 98.6 F)

After 1 week in the incubator, the honey water samples show small signs of mycellium growth, while the control sample remains negative on any growth.

TO DO:

1) Plate honey water samples onto agar to verify that growth in the medium is mycellium and not bacteria.

2) Once verified, use 30 mL of mycellium to knock up 4 lbs of rye that I have sitting in my desk. Is 30 mL enough or overkill?

3) Restock mycellium supply

4) Attempt transfection of cells with GFP ( this one is way out there, but what the hell, I have the facilities and reagents)

Reading between the lines, I would guess you can not turn down the temp on your incubator because the incubator is not yours and you are doing a little extra culturing while at work. If so, and if you like your job, you would be wise to incubate your cultures at YOUR room temp, buy a pressure cooker, and do all your 'brewing' at home.

LB media is for bacteria. I'd use something based on potato starch or something similar and get rid of the tryptone -- too much nitrogen, IMO. No salt, either. I have found that adding .5X MEM non-essential amino acid mix is benificial, btw. I just used this recipe and it worked great:

That should work as a broth too -- I don't know, I pretty much made the recipe up.

"After vortexing to break up the mycellium, the culture tube was incubated at 37 C ( 98.6 F)."

Yeah, lol -- what sylo said -- let me guess: you work in immunology? Yeah, someone would wonder what's up if an incubator was set at 29 in an immunology lab. Especially if growing a fungus that bruises blue. It will colonize faster, anyway, hidden somewhere at R.T.